Acetate toxicity tolerance in recombinant microbial host cells

Abstract

Acetate is a potent microbial inhibitor which can affect the performance of yeast in ethanolic fermentation. The present disclosure provides a recombinant microbial host cell having (i) a first genetic modification for increasing the activity of one or more proteins that function in a first metabolic pathway to convert acetate into an alcohol in the microbial host cell; (ii) a second genetic modification for increasing the activity of one or more proteins that function in a second metabolic pathway to import glycerol in the recombinant microbial host cell (iii) a third genetic modification for increasing the activity of one or more proteins that function in a third metabolic pathway to convert a C5 carbohydrate into ethanol in the microbial host cell. The recombinant microbial host cell comprises and natively expresses native proteins that function in a fourth native metabolic pathway to produce glycerol in the microbial host cell.

What is claimed is: 
     
       1. A recombinant microbial host cell comprising:
 a first genetic modification for increasing, when compared to a control microbial host cell lacking the first genetic modification, the activity of one or more proteins that function in a first metabolic pathway to convert acetate into an alcohol in the microbial host cell, wherein the one or more proteins that function in the first metabolic pathway comprise a protein having acetylating acetaldehyde dehydrogenase activity or a protein having acetylating acetaldehyde dehydrogenase activity and alcohol dehydrogenase activity (ADHE); 
 a second genetic modification for increasing, when compared to the control microbial host cell lacking the second genetic modification, the activity of one or more proteins that function in a second metabolic pathway to import glycerol in the recombinant microbial host cell, wherein the protein that function in the second metabolic pathway is an STL1 polypeptide; and 
 a third genetic modification for increasing, when compared to the control microbial host cell lacking the third genetic modification, the activity of one or more proteins that function in a third metabolic pathway to convert a C5 carbohydrate into the alcohol in the microbial host cell, wherein the one or more proteins that function in the third metabolic pathway comprise a protein having xylose isomerase activity from the genus  Catonella  sp.; and 
 wherein the recombinant microbial host cell comprises and natively expresses native proteins that function in a fourth native metabolic pathway to produce glycerol in the microbial host cell, wherein the native proteins that function in the fourth native metabolic pathway to produce glycerol comprise a GPD1 protein, a GPD2 protein, a GPP1 protein and a GPP2 protein. 
 
     
     
       2. The recombinant microbial host cell of  claim 1 , wherein the alcohol is ethanol. 
     
     
       3. The recombinant microbial host cell of  claim 1 , wherein the one or more proteins that function in the first, second or third metabolic pathway are heterologous proteins. 
     
     
       4. The recombinant microbial host cell of  claim 1 , wherein the one or more protein that function in the first metabolic pathway further comprises a protein having acetyl-CoA synthetase activity or is an ACS2 polypeptide. 
     
     
       5. The recombinant microbial host cell of  claim 1 , further comprising at least one of a first additional genetic modification, wherein the first additional genetic modification is:
 a deletion in at least one an aldose reductase gene; 
 a mutation in a polypeptide encoded by an iron-sulfur cluster gene; or 
 a mutation in a RAS2 polypeptide. 
 
     
     
       6. The recombinant microbial host cell of  claim 5 , wherein the aldose reductase gene is a GRE3 gene and/or a YPR1 gene. 
     
     
       7. The recombinant microbial host cell of  claim 5 , wherein the iron sulfur cluster gene is a YFH1 gene, a ISU1 gene and/or a NFS1 gene. 
     
     
       8. The recombinant microbial host cell of  claim 1  further comprising a fifth genetic modification for increasing the activity of one or more heterologous proteins that function in a fifth metabolic pathway for increasing the availability of electrons in the form of a reduced redox cofactor in the microbial host cell. 
     
     
       9. The recombinant microbial host cell of  claim 8 , wherein the fifth genetic modification is for increasing the activity of a NADPH-dependent alcohol dehydrogenase. 
     
     
       10. The recombinant microbial host cell of  claim 9 , wherein the NADPH-dependent alcohol dehydrogenase is an ADH1 polypeptide. 
     
     
       11. The recombinant microbial host cell of  claim 9 , further comprising a second additional genetic modification for increasing the activity of a protein capable of producing NADPH. 
     
     
       12. The recombinant microbial host cell of  claim 11 , wherein the protein capable of producing NADPH is at least one of a ZWF1 protein, a SOL3 protein and/or a GND1 protein. 
     
     
       13. The recombinant microbial host cell of  claim 1  which is a yeast host cell. 
     
     
       14. The recombinant microbial host cell of  claim 13  wherein the yeast host cell is a yeast of genus  Saccharomyces.    
     
     
       15. The recombinant microbial host cell of  claim 14 , wherein the yeast of genus  Saccharomyces  is a yeast of species  Saccharomyces cerevisiae.    
     
     
       16. The recombinant microbial host cell of  claim 1 , wherein the protein having acetylating acetaldehyde dehydrogenase and alcohol dehydrogenase activity is an ADHE polypeptide from the genus  Bifidobacterium  sp. or from  Bifidobacterium adolescentis.    
     
     
       17. The recombinant microbial host cell of  claim 4 , wherein the ACS2 polypeptide is from the genus  Saccharomyces  sp. or from  Saccharomyces cerevisiae.    
     
     
       18. The recombinant microbial host cell of  claim 1 , wherein the STL1 polypeptide is from the genus  Saccharomyces  sp. or  Pichia  sp.; or from  Saccharomyces cerevisiae  or  Pichia sorbitophila.    
     
     
       19. The recombinant microbial host cell of  claim 1 , wherein the protein having xylose isomerase activity is from  Catonella morbi.    
     
     
       20. The recombinant microbial host cell of  claim 1 , wherein the one or more proteins that function in the third metabolic pathway further comprise a protein having xylulokinase activity, a protein having transketolase activity, a protein having transaldolase activity, a protein having ribose-5-phosphate isomerase activity, and/or a protein having ribulose-phosphate 3-epimerase activity. 
     
     
       21. The recombinant microbial host cell of  claim 1 , wherein the one or more proteins that function in the third metabolic pathway further comprise an arabinose transporter, an ARAA polypeptide, an ARAB polypeptide, and/or an ARAD polypeptide. 
     
     
       22. The recombinant microbial host cell of  claim 21 , wherein at least one of the ARAA, ARAB or ARAD is from the genus  Bacteroides  sp. or from  Bacteroides  thetaiotaomicron.